Dual luciferase assay

Dual luciferase assay in using the Promega Dual luciferase assay:

Sample preparation:

• Freeze your leaf samples (4 leaf disks circa in 0,5 cm diameter) in liquid N₂ (in Eppendorf tubes with ~6 metal lysing beads) and keep them in liquid N₂ during transport. In the lab cool the Mixer Mill blocks in liquid nitrogen and then transfer your sample tubes into the holders. NOTE: Wear protective goggles or a visor when working with liquid N₂.
• Disrupt the leaf tissues by shaking for 1,5 min at 30 Hz frequency. After shaking refreeze your samples in liquid N₂.
• Tap the tubes firmly to gather powdered plant tissue to the bottom of the tube. Add 100 ml of 1 x passive lysis buffer and place tubes on ice. 
• Mix by vortexing for 10 s and spin at max speed for 5 min at 4 ^oC.
• Use 5 µl of the supernatant in the Dual luciferase reporter assay system (Promega): https://fi.promega.com/products/luciferase-assays/reporter-assays/dual_luciferase-reporter-assay-system/?catNum=E1910 

Notes:

Keep the samples on ice/at 4 oC after disruption and addition of the passive lysis buffer.

It is possible to conserve reagents by using eg. 1/2 of the volumes recommended in the protocol.

We measure chemiluminescence in white 96 well plates using a plate reader with injectors (samples are pipetted to the wells, and the addition of dual luciferase assay reagents and chemiluminescence readings are automated), but manual luminometers may also be used.